Single-Molecule Tracking in Live Cell without Immobilization or without Hydrodynamic Flow by Simulations: Thermodynamic Jitter^†

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

First, the format of this manuscript feels different from that of typical scientific papers. Furthermore, each sentence appears to require highly specialized knowledge. Much of the prerequisite knowledge seems to be based on the authors' past research, making it difficult to understand the significance of the research presented in this paper. Since my expertise does not entirely align with that of the authors, I will refrain from commenting on the importance of the paper in the context of their past research. However, I believe that many points in this manuscript need to be revised, as outlined below.

 

Major Points

1.While the vocabulary and sentences are highly specialized, there seems to be a lack of detailed explanations. It is difficult for readers without the prerequisite knowledge to understand.

2.The purpose of the paper is not clearly stated in the "Introduction." The novelty of the paper is also unclear.

3.The "Schemes" are presented as image data, making them difficult to understand. Especially for "Scheme 2," which explains equations, wouldn't it be better to include this directly in the text?

4.Out of the total 22 citations, 13 are self-citations. This raises doubts about whether the content of this paper is intended for a broad audience.

5.Two figures are presented in the results section, but it is not clear what is new compared to the authors' previous research. While quantitative data is shown in past papers by the authors, this paper only presents figures depicting the trajectories of two atoms and their statistical distributions, which seems to lower the quality of the paper. It gives the impression of being hastily prepared.

6.Considering the "self-citation," and the "low quality of the paper's format" as mentioned above, one cannot help but feel that the content of the paper itself appears to lack credibility.

 

 

Minor Points

1.In the beginning of the "Introduction," there is repetition of the same words.

2.At least the first two sentences seem to require citations.

3.There are unnatural highlights with a line marker in the text, giving the impression that the manuscript is still in draft form.

Comments on the Quality of English Language

Some sentences are difficult to understand. Plain explanation is required.

Author Response

Please find the responses attached. Thank you very much.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The article presents a significant advancement in single-molecule/particle biophysics and biochemistry through simulations demonstrating localized Brownian motion and fractal diffusion. The introduction and methods sections provide a thorough background and detailed methodology, although some areas could benefit from clearer explanations and smoother transitions. The results and discussion sections offer valuable insights but could be more engaging and accessible.  More information can be found in the Specific Comments below.

Specific Comments

Abstract

The abstract is clear and well-structured, presenting the motivation, key findings, and significance of the study. It outlines the challenge of measuring single molecules/particles at room temperature or under physiological conditions without immobilization or significant hydrodynamic flow. The mention of the novel simulation results demonstrating localized Brownian motion and fanning out in fractal diffusion is compelling. However, it could be improved by specifying the broader implications of these results for the field of single-molecule biophysics and biochemistry.

Introduction

The introduction provides a fair background on the importance of studying the transport and binding of individual molecules. The discussion on the limitations of current techniques and the relevance of fluorescence methods is well-articulated.

However, the flow could be improved by addressing redundancy and some awkward sentence structures, as well as:

• Line 30-31: Specify the main experimental technologies initially to avoid confusion.
• Line 58-63: Simplify the explanation of Poisson probability and its relevance.
• Line 70-75: Make the description of reentries and transitions more straightforward, or consider providing a visual aid.

Methods

The methods section is detailed, providing a clear explanation of the simulation approaches and the theoretical basis. It appropriately references prior work and lays out the steps taken in the study.

However, adding more context for specific terms and clarifying certain phrases would enhance readability:

• Line 106-107: Provide context for the generator "g24" from ref. [15].
• Line 114-115: Specify the disparities in the distribution function on the fractal structure.
• Line 116-118: Provide more context for what makes the investigated tracks typical.
• Line 120-121: Summarize the theoretical and simulation details from ref. [15].

Results

The results section acceptably presents the study's findings, highlighting the significance of single-molecule detection without immobilization. The comparison between Gaussian and gamma distributions is insightful.

However, the narrative could be made clearer and more engaging, and the rationale for choosing specific distributions should be better explained:

• Line 138-141: Simplify the explanation of thermodynamic jitter for broader accessibility. For example, “In this study, the concept of thermodynamic jitter was utilized to analyze the motion of single molecules within the cytoplasm of live cells. By simulating these movements and observing the resulting tracks, the researchers were able to demonstrate that individual molecules exhibit localized Brownian motion and fanning out patterns typical of fractal diffusion.”
• Line 146-149: Briefly summarize the criteria (criteria 1 to 3) mentioned in Scheme 1.
• Line 152-157: Provide a brief rationale for why the simulations are essential.
• Line 170-171: Explain the significance of the simulated diffusion tracks.
• Line 173-175: Provide more detail on why Gaussian and gamma distributions were chosen.
• Line 179-181: Make the comparison between Gaussian and gamma distributions more explicit in terms of their implications.

Discussion

The discussion provides a good analysis of the findings, connecting them to broader theoretical concepts. The importance of thermodynamic jitter and the implications for single-molecule studies are well-articulated.

However, the discussion starts abruptly and could benefit from a brief summary of key findings at the beginning. Please consider these adjustments:

• Line 219-221: Begin with a brief summary of key findings.
• Line 223-225: Provide context for why "Nℓmax = 32" is significant.
• Line 239-241: Provide concrete examples or applications for the discussion on thermodynamic signatures.
• Line 253-255: Make the explanation of measurement time more concise and directly tied to the study's findings.
• Line 258-265: Simplify the discussion of meaningful times to focus on the most critical points.
• Line 273-275: Tie the historical context directly to the study's contributions.
• Line 280-289: Summarize the discussion and highlight the study's broader implications more succinctly.

Conclusions

The conclusions succinctly summarize the main points of the study and suggest future research directions. Emphasizing the potential breakthrough in sensitivity for measuring individual molecules would strengthen this section. But a few items should be clarified:

• Line 293-295: Briefly summarize the formulas and relationships mentioned or keep them for the discussion section.
• Line 295-299: Directly link the call for increased sensitivity in measurements to the study's findings and potential future research directions.

Review's Concluding Remarks

After reviewing the study, I consider it meticulously conducted and has made a significant contribution to the field. However, some revisions are necessary to enhance the clarity and readability of the article. Addressing these points could greatly benefit the article and the readers.

Comments on the Quality of English Language

Language revisions required to improve the article's clarity and readability:

Introduction

• Line 24-26: Streamline the repetition of "Transport and binding of individual molecules."
• Line 27-29: Revise for readability, e.g., "There is broad consensus that analyzing these processes both in vitro and in vivo is crucial for life sciences."
• Line 36-37: Integrate the phrase "in a confocal fluorescence microscopy/spectroscopy setup" more smoothly.
• Line 41-43: Clarify what "them" refers to in "cannot be equated with them."
• Line 50-51: Integrate the mention of Markov processes more smoothly into the paragraph.
• Line 54-55: Avoid redundant referencing of Table 1.

Methods and Results

• Line 109-111: Clarify what "a limited continuous time random walk (LCTRW)" means.
• Line 130-133: Clarify the term "selfsame molecule/selfsame particle."
• Line 167-169: Revise for clarity the phrase "we have diffused to about 1 nanometer from another protein."

Discussion and Conclusion

• Line 229-230: Simplify the phrase "ensemble averaging in sparse subpopulations of single molecules."
• Line 233-235: Clarify "single-molecule/single-particle fingerprint of non-ergodicity."

Author Response

Please find the responses attached. Thank you very much.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The revisions have been made appropriately following the comments. While the current version is already at a publishable standard, I am concerned that the number of self-citations exceeds 50% of the total. I believe it would improve the paper to include more references to other studies.